Tropomyosin has generally been considered to be a rigid rod in the regulation of muscle contraction. There is information, however, from this and other laboratories to indicate that tropomyosin is flexible and that the carboxyl-terminal half of the molecule is more flexible than the amino-terminal half. Our more recent data have also shown that the conformation in the carboxyl-terminal half of tropomyosin is more sensitive to the interaction with the other regulatory proteins and calcium, suggesting that these conformational changes in tropomyosin are important to its role in the regulation of contraction. Our aims are to learn about the role of the conformational changes in regulation by mapping out regions of structural change within tropomyosin during its interaction with other muscle proteins in reconstituted contractile systems. We will pursue these aims with nitroxide spin probes selectively attached to cysteines 190 and 36 in the carboxyl- and amino-terminal halves, respectively, of tropomyosin. First of all, conformational states within tropomyosin alone will be investigated under a variety of conditions by measuring changes in mobility and mobility states of the attached spin labels. We will then measure changes in label mobility when tropomyosin interacts with other thin filament proteins and calcium to understand the molecular mechanisms whereby calcium switches tropomyosin from the inhibiting to acitivating states. Myosin will then be added to the reconstituted thin filament with to understand the recently postulated role of myosin in controlling tropomyosin's regulatory role.